U.S. patent application number 11/631729 was filed with the patent office on 2008-09-11 for transgenic non-human animal for use in research models for studying parkinson's disease.
Invention is credited to Mats Ekstrand, Nils-Goran Larsson, Lars Olson.
Application Number | 20080222738 11/631729 |
Document ID | / |
Family ID | 35783183 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080222738 |
Kind Code |
A1 |
Larsson; Nils-Goran ; et
al. |
September 11, 2008 |
Transgenic Non-Human Animal for Use in Research Models for Studying
Parkinson's Disease
Abstract
The invention relates to a transgenic mouse model with deficient
respiratory chain function in dopamine (DA) neurons. By suppressing
or deleting the mitochondrial transcription factor A (Tfam) in DA
neurons, a mouse model is obtained, which reproduces key
pathophysiological features of Parkinson's disease (PD), i.e., slow
progressive loss of DA terminals in striatum and loss of DA neurons
in substantia nigra pars compacta; alpha-synuclein immunoreactivity
including intracellular inclusions similar to Lewy bodies in
affected areas prior to and during cell loss; progressive movement
disorder associated with abnormal gait, tremor and rigid limbs. The
mouse model can be used to develop pharmacological, gene therapy or
cell therapy treatments for PD.
Inventors: |
Larsson; Nils-Goran;
(Huddinge, SE) ; Ekstrand; Mats; (Huddinge,
SE) ; Olson; Lars; (Huddinge, SE) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,;KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Family ID: |
35783183 |
Appl. No.: |
11/631729 |
Filed: |
July 7, 2005 |
PCT Filed: |
July 7, 2005 |
PCT NO: |
PCT/SE05/01136 |
371 Date: |
December 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60521813 |
Jul 7, 2004 |
|
|
|
Current U.S.
Class: |
800/3 ; 800/18;
800/25 |
Current CPC
Class: |
A01K 2267/0306 20130101;
A01K 2227/105 20130101; A01K 67/0276 20130101; A01K 2217/075
20130101; C07K 14/4702 20130101; C12N 2800/30 20130101; C12N
15/8509 20130101 |
Class at
Publication: |
800/3 ; 800/18;
800/25 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C12N 15/89 20060101 C12N015/89; A01K 67/027 20060101
A01K067/027 |
Claims
1. A transgenic non-human mammal genetically modified to have
respiratory chain dysfunction or genetically modified to lack
respiratory chain function in dopaminergic (DA) neurons only.
2. A transgenic non-human mammal according to claim 1,
characterised in that said mammal is a rodent, preferably a rat or
a mouse.
3. A transgenic non-human animal according to claim 1,
characterised in that said animal is genetically modified to lack
oxidative phosphorylation function in dopaminergic (DA) neurons
only, or to have oxidative phosphorylation dysfunction in
dopaminergic (DA) neurons only.
4. A transgenic non-human animal according to claim 3,
characterised in that mitochondrial transcription factor A (Tfam)
has been selectively suppressed or deleted in dopaminergic (DA)
neurons.
5. A method for producing a transgenic non-human animal according
to anyone of claims 1-4, comprising the steps of: a) providing a
first group of non-human animals, said first group of non-human
animals being homozygous for a loxP-flanked Tfam allele
(Tfam.sup.loxP/Tfam.sup.loxP); b) providing a targeting vector
comprising a NLS-cre gene inserted into the dopamine transporter
locus; c) introducing said targeting vector into embryonic stem
cells originating from said non-human animal by homologous
recombination; d) growing said recombinant embryonic stem cells
according to known methods thereby obtaining a transcenic non-human
animal expressing cre in dopaminergic (DA) neurons; e) crossing
said first group of non-human animals being homozygous for a
loxP-flanked Tfam allele (Tfam.sup.loxP/Tfam.sup.loxP) with said
non-human animal expressing cre in dopaminergic (DA) neurons,
thereby obtaining a transgenic non-human mammal genetically
modified to have respiratory chain dysfunction or genetically
modified to lack respiratory chain function in dopaminergic (DA)
neurons only.
6. A method for investigating whether a chemical compound could be
used for treating Parkinson's disease, comprising the steps of a)
providing a transgenic non-human animal according to anyone of
claims 1-4; b) providing a chemical compound to be tested; c)
exposing said transgenic non-human animal to said compound to be
tested; d) controlling whether said chemical compound to be tested
affects locomotion and/or rearing of the transgenic non-human
animals, which indicates that said compound could be used for
treating Parkinson's disease.
7. Use of a compound for preparing a pharmaceutical composition for
treating Parkinson's disease, wherein said compound has been shown
to be useful for treating Parkinson's disease by the method
according to claim 6.
Description
FIELD OF THE INVENTION
[0001] The invention relates to research models for studying
disease. Specifically, the invention relates to research models for
studying Parkinson's disease. Even more specifically, the invention
relates to a transgenic non-human animal, in particular a mouse,
that can be used as a model system when studying Parkinson's
disease. The invention also provides screening methods aiming at
finding and identifying pharmaceuticals, therapies and treatments
for Parkinson's disease.
BACKGROUND AND SUMMARY OF THE INVENTION
[0002] Cardinal symptoms in Parkinson's disease (PD) result
primarily from death of dopaminergic (DA) neurons in substantia
nigra pars compacta (SNpc) (Dauer et al., Neuron 39, 889-909
(2003); Dawson et al., Science 302, 819-22 (2003)). Current
interventions ameliorate symptoms but there is no treatment to halt
or delay loss of DA neurons. Several lines of evidence suggest that
respiratory chain dysfunction is involved in the pathogenesis of PD
(Dauer et al., Neuron 39, 889-909 (2003); Dawson et al., Science
302, 819-22 (2003)): (1) A deficient function of the respiratory
chain has been reported in cell lines and tissues from PD patients
(Mizuno, Y. et al., Bioch. Biophys. Res. Comm. 163, 1450-1455
(1989); Schapira, Adv Neurol 86, 155-62 (2001)). (2) Certain
mitochondrial DNA (mtDNA) polymorphisms reduce the risk of PD (van
der Walt, et al., Am J Hum Genet 72, 804-11 (2003)). (3) The toxin
MPP+ inhibits complex I of the respiratory chain and it causes
parkinsonism after selective uptake in DA neurons (Langston et al.,
Science 219, 979-80 (1983); Mizuno et al., J Neurochem 48, 1787-93
(1987)). (4) The pesticide rotenone is a strong inhibitor of
complex I and long term exposure causes parkinsonism in rats
(Betarbet et al., Nat Neurosci 3, 1301-6 (2000)). (5) The herbicide
paraquat inhibits complex I and can in combination with other
agents cause selective degeneration of DA neurons (Thiruchelvam et
al., J Neurosci 20, 9207-14 (2000)). In addition, the mitochondrial
respiratory chain is a major producer of ROS and oxidative stress
is directly involved in the cascade of biochemical changes causing
DA cell death (Thiruchelvam et al., J Neurosci 20, 9207-14
(2000)).
[0003] The respiratory chain consists of five enzyme complexes
formed by .about.100 different protein subunits that perform the
process of oxidative phosphorylation to generate adenosine
triphosphate (ATP), which is used as the energy source for a large
number of cellular processes (Saraste, Science 283, 1488-93
(1999)). The biogenesis of the respiratory chain is unique in its
bipartite dependence on both nuclear and mtDNA genes. The
expression and maintenance of mtDNA is completely controlled by
nuclear genes, as exemplified by mitochondrial transcription factor
A (TFAM) which regulates mtDNA transcription (Fisher et al., J.
Biol. Chem. 260, 11330-11338 (1985); Parisi et al., Science 252,
965-969 (1991); Larsson et al., Annu. Rev. Genet. 29, 151-178
(1995); Falkenberg et al. Nat. Genet. 31, 289-294 (2002)) and mtDNA
copy number (Ekstrand et al., Hum. Mol. Genet. 13, 935-944 (2004)).
We have constructed a conditional knockout allele (Gu et al.,
Science 265, 103-106 (1994)) by inserting loxP sequences into the
Tfam locus to generate Tfam.sup.loxP animals (Larsson et al., Nat.
Genet. 18, 231-6 (1998)). The Tfam.sup.loxP mice have normal TFAM
protein expression and normal oxidative phosphorylation capacity
(Wang et al., Nat. Genet. 21, 133-7 (1999)). The protein cre
recombinase will specifically recognize and recombine loxP
sequences, thereby deleting any DNA between them. By controlling
the expression of cre we can in our system choose in what tissue or
cell type we want to knock out Tfam. We have extensively documented
that this system allows very efficient germ line and
tissue-specific knockout of Tfam (Larsson et al., Nat. Genet. 18,
231-6 (1998); Wang et al., Nat. Genet. 21, 133-7 (1999); Li et al.,
Proc. Natl. Acad. Sci. USA 97, 3467-72 (2000); Hansson et al., Proc
Natl Acad Sci USA 101, 3136-3141 (2004); Silva et al., Nat. Genet.
26, 336-340 (2000); Sorensen et al., J. Neurosci. 21, 8082-8090
(2001); Wredenberg et al., Proc Natl Acad Sci USA 99, 15066-71
(2002)). Heterozygous germ line knockout animals (+/Tfam.sup.-)
have a reduction of mtDNA copy number in all tissues and a moderate
respiratory chain deficiency in the heart, whereas homozygous germ
line knockouts (Tfam.sup.-/Tfam.sup.-) die in mid-gestation due to
lack of mtDNA and absence of oxidative phosphorylation (Larsson et
al., Nat. Genet. 18, 231-6 (1998)). We have also studied
pathophysiological events associated with mitochondrial dysfunction
by selectively disrupting Tfam in heart muscle cells (Wang et al.,
Nat. Genet. 21, 133-7 (1999); Li et al., Proc. Natl. Acad Sci. USA
97, 3467-72 (2000); Hansson et al., Proc Natl Acad Sci USA 101,
3136-3141 (2004)), insulin-secreting .beta.-cells (Silva et al.,
Nat. Genet. 26, 336-340 (2000)), forebrain neurons (Sorensen et
al., J. Neurosci. 21, 8082-8090 (2001)) and skeletal muscle cells
(Wredenberg et al., Proc Natl Acad Sci U S A 99, 15066-71
(2002)).
[0004] A critical gap in our knowledge of PD is a validated and
widely acceptable rodent model in which endogenous disruption of
respiratory enzymes leads to a PD-like phenotype. An ideal genetic
model for PD would have the following characteristics (Beal, Nat
Rev Neurosci 2, 325-34 (2001)): (1) Normal number of DA neurons at
birth with a gradual loss during adulthood. (2) Easily detectable
motor deficits including bradykinesia, rigidity and resting tremor.
(3) Histology showing typical .alpha.-synuclein pathology including
formation of the intracellular inclusions known as Lewy bodies. (4)
Robust genetics allowing easy propagation of the genotype. (5) A
progressive disease course of just a few months for rapid testing
of different therapeutic strategies.
[0005] Almost all available in vivo models of PD are based on the
administration of neurotoxins to animals. The first agent used to
produce an animal model of PD was 6-OHDA (Ungerstedt, Acta Physiol
Scand Suppl 367, 69-93 (1971)). This toxin is normally injected
unilaterally directly into the substantia nigra, the medial
forebrain bundle or the striatum causing a selective loss of DA
neurons. 6-OHDA lesions do not result in typical Lewy body
formation and can cause nonspecific damage to other neurons. When
injected into the substantia nigra 6-OHDA treatment is acute,
causing a rapid cell loss not resembling the progressive nature of
PD. If injected into striatum the effect is slower (<4 weeks)
but then only partial. This limits researchers to study only the
end-stage or a very short progression of the disease (Beal et al.,
Nat Rev Neurosci 2, 325-34 (2001); Ungerstedt, Acta Physiol Scand
Suppl 367, 69-93 (1971); Deumens et al., Exp Neurol 175, 303-17
(2002)).
[0006] The most widely used animal model of PD today is acute
systemic administration of MPTP which in primates causes
parkinsonism with a selective loss of DA neurons in the substantia
nigra. Rodents are much less sensitive to MPTP and require a higher
dose while the typical behavioral symptoms of parkinsonism rarely
appear. The main disadvantages of MPTP is the resistance of most
rodents and the acute nature of the model preventing researchers
from studying the progression of the disease (Shimohama et al.,
Trends Mol Med 9, 360-5 (2003)).
[0007] Recently it was discovered that systemic treatment with low
doses of the naturally occurring pesticide rotenone over time
causes parkinsonism in rats. Treated animals show a selective loss
of DA neurons in the substantia nigra associated with intracellular
inclusions similar to Lewy bodies. The outcome of rotenone
treatment is highly variable as only a part of the treated rats
develop PD-like pathology and some strains do not respond at all to
the drug (Betarbet et al., Nat Neurosci 3, 1301-6 (2000)). All
these pharmacological models also suffer from the inherent risk of
other pleiotropic effects of systemic drug administration.
[0008] In the present document we describe the generation of a
mouse with a specific disruption of Tfam and hence oxidative
phosphorylation in DA neurons only. We use the dopamine transporter
(DAT) locus to direct the expression of cre recombinase to this
specific cell type. Such mice over the time of several months
develop a PD-like behavioral phenotype associated with loss of DA
neurons in the SNpc and pathology typical for the disease.
[0009] The generated animal model faithfully reproduces key
pathophysiological features of PD, i.e. slow progressive loss of DA
terminals in striatum and loss of DA neurons in SNpc;
.alpha.-synuclein reactivity including intracellular inclusions
similar to Lewy bodies in affected areas prior to and during cell
loss; progressive movement disorder, that is partially reversed by
L-DOPA treatment, associated with abnormal gait, tremor and rigid
limbs. This model will be a valuable tool for testing
pharmacological, gene and cell therapies to counteract or cure
PD.
[0010] As used herein, the term "genetically modified" refers to
any purposeful alteration of the naturally-occurring genome of an
animal. For example, sequences can be inserted into the genome
which, when activated, can cause selective deactivation of a
particular gene adjacent to the inserted sequence.
[0011] The term "dysfunction" refers to any deviation from normal
or naturally-occurring function in a disease-free state.
[0012] The term "naturally exhibiting" refers to the
characteristics or traits occurring under normal environmental and
physiological conditions.
[0013] As used herein, the term "Tfam" relates to mitochondrial
transcription factor A (Fisher et al., J. Biol. Chem. 260,
11330-11338 (1985); Parisi et al., Science 252, 965-969 (1991);
Larsson et al., Annu. Rev. Genet. 29, 151-178 (1995); Falkenberg et
al. Nat. Genet. 31, 289-294 (2002)) and mtDNA copy number (Ekstrand
et al., Hum. Mol. Genet. 13, 935-944 (2004)).
[0014] As disclosed herein, the terms "Parkinson mouse" and
"homozygous knockout animals" both relate to transgenic mice
comprising the 5' end of the dopamine transporter gene fused to a
DNA sequence encoding cre recombinase as well as a DNA sequence
comprising a sequence encoding Tfam which sequence also contains
LoxP sequences (+/DAT-cre; Tfam.sup.loxP/Tfam.sup.loxP). This
recombination leads to cell type specific homozygous disruption of
Tfam and hence oxidative phosphorylation in DA neurons only, and
accordingly such mice display symptoms of Parkinson's disease.
SUMMARY OF THE INVENTION
[0015] In a first aspect, the present invention relates to a
transgenic non-human mammal genetically modified to have
respiratory chain dysfunction or genetically modified to lack
respiratory chain function in dopaminergic (DA) neurons only. In a
preferred embodiment, said mammal is a rodent, preferably a rat or
a mouse.
[0016] Furthermore, it is preferred that said animal is genetically
modified to lack oxidative phosphorylation function in dopaminergic
(DA) neurons only, or to have oxidative phosphorylation dysfunction
in dopaminergic (DA) neurons only. This can be done by selectively
suppressing or deleting mitochondrial transcription factor A (Tfam)
in dopaminergic (DA) neurons
[0017] In a second aspect, the present invention provides a method
for producing a transgenic non-human animal comprising the steps
of:
a) providing a first group of non-human animals, said first group
of non-human animals being homozygous for a loxP-flanked Tfam
allele (Tfam.sup.loxP/Tfam.sup.loxP); b) providing a targeting
vector comprising a NLS-cre gene inserted into the dopamine
transporter locus; c) introducing said targeting vector into
embryonic stem cells originating from said non-human animal by
homologous recombination; d) growing said recombinant embryonic
stem cells according to known methods thereby obtaining a
transcenic non-human animal expressing cre in dopaminergic (DA)
neurons; e) crossing said first group of non-human animals being
homozygous for a loxP-flanked Tfam allele
(Tfam.sup.loxP/Tfam.sup.loxP) with said non-human animal expressing
cre in dopaminergic (DA) neurons, thereby obtaining a transgenic
non-human mammal genetically modified to have respiratory chain
dysfunction or genetically modified to lack respiratory chain
function in dopaminergic (DA) neurons only.
[0018] In a third aspect, the present invention relates to a method
for investigating whether a chemical compound could be used for
treating Parkinson's disease, comprising the steps of
a) providing a transgenic non-human animal according to anyone of
claims 1-4; b) providing a chemical compound to be tested; c)
exposing said transgenic non-human animal to said compound to be
tested; d) controlling whether said chemical compound to be tested
affects locomotion and/or rearing of the transgenic non-human
animals, which indicates that said compound could be used for
treating Parkinson's disease.
[0019] "Amino acid sequence", "polypeptide sequence" and "peptide
sequence" are used interchangeably herein to refer to a sequence of
amino acids.
[0020] Nucleic acid molecule", "nucleic acid sequence", "nucleotide
sequence" and "polynucleotide sequence" as used herein refer to an
oligonucleotide or polynucleotide, and fragments or portions
thereof, and to DNA or RNA of genomic or synthetic origin which may
be single- or double-stranded, and represent the sense or antisense
strand.
[0021] As used herein, the terms "oligonucleotides" and "oligomers"
refer to a nucleic acid sequence of at least about 10 nucleotides
and as many as about 60 nucleotides, preferably about 15 to 30
nucleotides, and more preferably about 20-25 nucleotides, which can
be used as a probe or amplimer.
[0022] As used herein, the terms "complementary" or
"complementarity" are used in reference to "polynucleotides" and
"oligonucleotides" (which are interchangeable terms that refer to a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "5'-CAGT-3'," is complementary to the
sequence "5'-ACTG-3'." Complementarity can be "partial" or "total."
"Partial" complementarity is where one or more nucleic acid bases
is not matched according to the base pairing rules. "Total" or
"complete" complementarity between nucleic acids is where each and
every nucleic acid base is matched with another base under the base
pairing rules. The degree of complementarity between nucleic acid
strands has significant effects on the efficiency and strength of
hybridization between nucleic acid strands. This is of particular
importance in amplification reactions, as well as detection methods
which depend upon binding between nucleic acids.
[0023] As used herein, the term "an oligonucleotide having a
nucleotide sequence encoding a gene" means a nucleic acid sequence
comprising the coding region of a gene, i.e. the nucleic acid
sequence which encodes a gene product. The coding region may be
present in either a cDNA, genomic DNA or RNA form. When present in
a DNA form, the oligonucleotide may be single-stranded (i.e., the
sense strand) or double-stranded. Suitable control elements such as
enhancers, promoters, splice junctions, polyadenylation signals,
etc. may be placed in close proximity to the coding region of the
gene if needed to permit proper initiation of transcription and/or
correct processing of the primary RNA transcript. Alternatively,
the coding region utilized in the expression vectors of the present
invention may contain endogenous enhancers, splice junctions,
intervening sequences, polyadenylation signals, etc. or a
combination of both endogenous and exogenous control elements.
[0024] Vectors are frequently used in the experiments disclosed in
the present application. A suitable vector can be a plasmid,
cosmid, phage, etc. Vectors are standard tools of molecular
biologists (J. Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY, US, pp
9.31-9.58).
[0025] A "selection marker" as used herein refers to a nucleic acid
sequence which is used for facilitating screening of successful
transformants. Examples of selection markers are antibiotic
resistance genes, enzyme genes, etc. Selection markers are standard
tools of molecular biologists (J. Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY,
US).
[0026] A "transgenic animal" as used herein refers to an animal
that includes a transgene which is inserted into a cell and which
becomes integrated into the genome either of somatic and/or germ
line cells of the. A "transgene" means a DNA sequence which is
partly or entirely heterologous (i.e., not present in nature) to
the animal in which it is found, or which is homologous to an
endogenous sequence (i.e., a sequence that is found in the animal
in nature) and is inserted into the animal's genome at a location
which differs from that of the naturally occurring sequence.
Transgenic animals which include one or more transgenes are within
the scope of this invention.
[0027] The term "transfection" as used herein refers to the
introduction of foreign DNA into cells. Transfection may be
accomplished by a variety of means known to the art including
calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, biolistics (i.e. particle bombardment) and
the like.
[0028] The term "compound" refers to any chemical entity,
pharmaceutical, drug, and the like that is suspected of having a
positive impact on Parkinson's disease. Compounds comprise both
known and potential therapeutic compounds. A compound can be
considered to positively affect Parkinson's disease if it can be
shown that locomotion and rearing of the transgenic non-human
mammal increases after exposure to said compound in accordance with
behavioral analysis as outlined below.
[0029] A compound is said to be "in a form suitable for
administration such that the compound is bio-available in the blood
of the animal" when the compound may be administered to an animal
by any desired route (e.g., oral, intravenous, subcutaneous,
intrathecal, intraperitoneal, intramuscular, etc.) and the compound
or its active metabolites appears in the blood of the animal in an
active form.
[0030] Following initial screening, a compound that appears
promising is further evaluated by administering various
concentrations of the compound to the transgenic animals provided
herein in order to determine an approximate therapeutic dosing
range.
[0031] Animal testing may be supplemented and confirmed by testing
on human subjects. However, the animal models herein provided allow
the testing of a large number of compounds, both by the methods
described above and other methods known in the art, in a system
similar in many important respects to that in humans.
[0032] "Amplification" is defined as the production of additional
copies of a nucleic acid sequence and is generally carried out
using polymerase chain reaction technologies well known in the art
(Dieffenbach C W and G S Dveksler (1995) PCR Primer, a Laboratory
Manual, Cold Spring Harbor Press, Plainview N.Y.). As used herein,
the term "polymerase chain reaction" ("PCR") refers to the method
of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby
incorporated by reference, which describe a method for increasing
the concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. The length of the
amplified segment of the desired target sequence is determined by
the relative positions of two oligonucleotide primers with respect
to each other, and therefore, this length is a controllable
parameter. By virtue of the repeating aspect of the process, the
method is referred to as the "polymerase chain reaction"
(hereinafter "PCR"). Because the desired amplified segments of the
target sequence become the predominant sequences (in terms of
concentration) in the mixture, they are said to be "PCR
amplified."
[0033] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (e.g., hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of
.sup.32P-labeled deoxynucleotide triphosphates, such as dCTP or
dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide sequence can be amplified with the appropriate set
of primer molecules. In particular, the amplified segments created
by the PCR process itself are, themselves, efficient templates for
subsequent PCR amplifications.
[0034] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and of an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0035] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labelled with any "reporter molecule", so that it
is detectable in any detection system, including, but not limited
to enzyme (e.g., ELISA, as well as enzyme-based histochemical
assays), fluorescent, radioactive, and luminescent systems. It is
not intended that the present invention be limited to any
particular detection system or label.
[0036] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double- or single-stranded DNA at or near a specific nucleotide
sequence.
[0037] The term "Southern blot" refers to the analysis of DNA on
agarose or acrylamide gels to fractionate the DNA according to
size, followed by transfer and immobilization of the DNA from the
gel to a solid support, such as nitrocellulose or a nylon membrane.
The immobilized DNA is then probed with a labeled
oligo-deoxyribonucleotide probe or DNA probe to detect DNA species
complementary to the probe used. The DNA may be cleaved with
restriction enzymes prior to electrophoresis. Following
electrophoresis, the DNA may be partially depurinated and denatured
prior to or during transfer to the solid support. Southern blots
are a standard tool of molecular biologists (J. Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, NY, pp 9.31-9.58).
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1: Knock-in construct and genotyping:
[0039] A. A cassette containing an NLS-cre (cre) gene followed by
an FRT-flanked neomycin (neo) gene was inserted in the FspI site
just upstream of Exon 2. The Intron 1/Exon 2 border was recreated
by introducing a short oligo 5' to the NLS-cre gene. Homologous
recombination in ES-cells generated the DAT-cre.sup.neo locus. The
neo gene was later removed by breeding to FLPe deleter mice. B.
Southern blot genotyping of BamHI digested genomic DNA. The
hybridization probe located outside the area of homology gives
bands of different sizes for each of the three DAT loci. C. Routine
PCR genotyping of genomic tail DNA. Unspecific recombination of the
Tfam locus in tail DNA is not detectable when the neo gene has been
removed from the knockin locus. The Tfam locus was genotyped using
a three primer protocol that gives specific bands for the wild-type
(wt; 404 bp), loxP-flanked (Tfam.sup.loxP; 437 bp) and knockout
(Tfam.sup.KO; 329 bp) Tfam loci. The presence of either cre knockin
loci (DAT-cre.sup.neo or DAT-cre) was demonstrated by using PCR
primers specific for the cre gene (371 bp). PCR primers spanning
the neo gene were used to discriminate between the DAT-cre.sup.neo
and DAT-cre loci. These primers only give a product in the absence
of neo.
[0040] FIG. 2: Analyses of spontaneous activity and motor
function:
[0041] The locomotion (A-C) and rearing (D-F) activities of
homozygous knockout and control mice were measured for 60 minutes
in open-field activity cages. The 10 week old homozygous knockout
mice did not behave significantly different from age-matched
controls (A and D) while 14 week old homozygous knockout mice
displayed decreased activity (B and E). Total locomotion (C) and
rearing (F) during 60 minutes decreased further in aging homozygous
knockout mice, as compared to age-matched controls. G-H. Rota-Rod
was used to assess motor function in homozygous knockout mice. 10
week old homozygous knockout mice did not perform significantly
different from controls (G) while 14 week old mice performed
significantly different from control mice during the second and
third trials (H). I. Mean weight of male homozygous knockout and
control mice at 16, 20 and 30 weeks of age. Error bars are
indicated as .+-.S.E.M. Statistically significant differences to
age-matched controls are indicated as: *p<0.05; **p<0.01;
***p<0.001.
[0042] FIG. 3: Histochemical and biochemical analyses of the
midbrain DA system.
[0043] A-F. TH immunoreactivity in striatum of control (A-B) and
homozygous knockout (C-F) mice. Loss of DA terminals starts in
dorsolateral caudate putamen (CPu) and progress to affect also more
medial and ventral areas of striatum (D-F). In old (43 weeks)
homozygous knockout mice only part of nucleus accumbens (NAcc)
contain TH immunoreactive fibers (F). Bar=500 .mu.m. G-L. TH
immunoreactivity in substantia nigra pars compacta (SNpc) and the
ventral tegmental area (VTA) of control (G-H) and homozygous
knockout (I-L) mice. Loss of DA neurons is first seen in SNpc but
then progresses to involve also VTA (J-L). Bar=400 .mu.m. M-N. High
magnification of TH immunoreactive nerve terminals in striatum of 6
and 43 week old homozygous knockout mice. Bar=10 .mu.m. O-P. HPLC
measurements of DA levels in striatum (0) as well as other brain
regions (P) of 20 week old control and homozygous knockout mice. Q.
DA levels related to the DA metabolites HVA or DOPAC as an
estimation of DA turnover in striatum of 20 week old control and
homozygous knockout mice. Error bars are indicated as .+-.S.E.M.
Statistically significant differences to age-matched controls are
indicated as: *p<0.05; **p<0.01; ***p<0.001.
[0044] FIG. 4; Formation of intra-cytoplasmic inclusions and L-DOPA
treatment.
[0045] A and B. Confocal microscopic demonstration of
.alpha.-synuclein immunoreactive inclusions (green) in TH
immunoreactive DA neurons (red) of the ventral tegmental area (VTA)
and substantia nigra (SN). C. Example of triple co-localization of
TH immunoreactivity (blue), .alpha.-synuclein immunoreactivity
(green) and ubiquitin immunoreactivity (red) in a midbrain DA
neuron of a homozygous knockout mouse. The neuropil of homozygous
knockout and control mice contain finely dispersed ubiquitin
immunoreactivity. In addition, larger ubiquitin immunoreactive
aggregates that also are .alpha.-synuclein immunoreactive can be
found in the affected DA neurons of homozygous knockout mice. This
is seen projected in three planes in the lower right of the three
TH immunoreactive neurons (yellow spot). D. Small somata of the
tubero-infundibular dopamine system (A12) are TH immunoreactive and
do not show any obvious neuropathological changes in 43 weeks old
homozygous knockout mice (scale bar: 25 .mu.m). E-H. Cohorts of 20
and 30 week old homozygous knockout or control mice were treated
with L-DOPA (20 mg/kg) or saline. Homozygous knockout mice of both
ages responded to L-DOPA treatment with increased locomotion (E)
and rearing (G). L-DOPA treatment of younger homozygous knockout
mice resulted in a greater locomotion response than treatment of
older homozygous knockout mice (E-F). The increase of rearing after
L-DOPA treatment of homozygous knockout mice was similar in both
age groups (G-H). Error bars are indicated as .+-.S.E.M. The bars
show mean locomotion (E) and mean rearing (G) in control and
homozygous knockout mice treated with saline (sal) or L-DOPA (LD).
Statistically significant differences are indicated with stars:
*p<0.05; **p<0.01; ***p<0.001.
[0046] FIG. 5: Dopamine neuron specific expression of cre
transgene
[0047] A. In situ hybridization of midbrain dopamine neurons.
Tyrosine hydroxylase (TH) and cre recombinase specific
.sup.33P-labeled hybridization probes were used to estimate the
specificity of cre expression. B. LacZ reporter mice visualization
of cre expression. Mice carrying the DAT-cre knockin locus were
mated to ROSA26-R reporter mice. Offspring heterozygous for both
loci were identified and studied with immunohistochemistry. TH and
.beta.-galactosidase (.beta.-gal) specific polyclonal antibodies
were used to visualize these proteins in brain sections.
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Generation of DAT-cre Mice
[0048] In order to obtain mouse genomic DNA clones containing the
5' end of the dopamine transporter gene (DAT; Solute carrier family
6, member 3) we screened a 129/SvJ .lamda. Fix II phage library
(Stratagene) using an 814 bp probe spanning from the promoter
region over exon 1 and into intron 1. The probe was generated by
PCR using mouse genomic DNA with the forward primer 5'-CAG GGT CGG
AGA GTC ATA CAA C-3' (SEQ ID NO 10) and the reverse primer 5'-ATG
AAC CAG GTC TTG AGT CTG G-3' (SEQ ID NO 11). Five overlapping
clones containing part of the DAT gene were identified and
isolated. Restriction enzyme digestion followed by Southern
blotting and hybridization with oligo probes was used to make an
approximate contig of the five clones. Clone 1, spanning from
.about.3.7 kb upstream of exon 1 and downstream into intron 5, was
released with NotI and subcloned into pBlueScript II SK, generating
pME1.
[0049] An NLS-cre/FRT-neo-FRT cassette was constructed by
subcloning the needed elements in several steps. First the MCS of a
pBlueScript II SK was released with SalI-KpnI and replaced with a
synthetic oligo containing the necessary cloning sites,
inactivating the 3' KpnI site
(SacI/PacI/SalI/EcoRI/KpnI/FRT-sequence/XhoI/EcoRI/SacII/AscI/KpnI*),
generating pME2. An NLS-cre fragment was released from pML78 (Mark
B. Lewandoski, National Cancer Institute at Frederick) using
SalI-KpnI and ligated into pME2, generating pME3. Next, a synthetic
oligo containing an FRT-sequence was introduced after the polyA in
pMC1neo-polyA (provided by Thomas Perlmann, Karolinska Institute),
using BamHI and SacII. This neo-FRT construct was then released
with XhoI-SacII and ligated into pME3, generating pME4 which was
sequenced from both directions.
[0050] For final assembly of the targeting construct a new plasmid
was generated by replacing the MCS of pBlueScript II SK with an
oligo containing restriction sites for the insertion of 5' and 3'
homology arms as well as for the NLS-cre/FRT-neo-FRT cassette
(SacI/NotI/BamHI/SmaI/PacI/BamHI/AscI/EcoRV/BamHI/EcoRI/KpnI),
generating pME5. The 5' and 3' homology arms were released from
pME1 with NotI-FspI and FspI-EcoRI, respectively, and inserted into
pME5 using NotI-SmaI and EcoRV-EcoRI. The cassette from pME4 was
released and inserted between the homology arms using PacI-AscI.
The endogenous DAT translational start site is located in exon 2
and since this design actually puts the cassette in intron 1 just
before exon 2 we finally introduced a short synthetic oligo
recreating the intron 1/exon 2 border at the PacI site just
upstream of the cre gene, generating pME6. The insertion site was
sequenced to verify the correct integration and direction of the
oligo.
[0051] The targeting vector was linearized with NotI and
electroporated into RI embryonic stem (ES) cells. Candidate
recombinant clones were selected for by growth in the presence of
G418. Southern hybridization showed a .about.30% targeting
efficiency of the DAT-locus with the knockin construct. Three
correctly targeted clones were injected into C57BL/6J blastocysts
which were then implanted into pseudopregnant females. Two of the
clones generated medium- to high-grade male chimeras that gave rise
to agouti pups when bred to C57BL/6J females, indicating germline
transmission of the ES-cell genome containing the targeted
DAT-locus. Agouti pups were genotyped using Southern blot as well
as PCR.
[0052] To remove the FRT-flanked neo gene downstream of the cre
gene, mice heterozygous for the targeted DAT-locus
(+/DAT-cre.sup.neo) were mated to FLPe deleter mice, expressing
FLPe from a human .beta.-actin promoter. Offspring lacking the neo
gene (+/DAT-cre) were identified with Southern blot analysis and
kept on a C57BL/6J background.
Generating Parkinson Mice
[0053] DAT-cre mice were mated to mice homozygous for a
loxP-flanked mitochondrial transcription factor A (Tfam) allele
(Tfam.sup.loxP/Tfam.sup.loxP) and offspring carrying the DAT-cre
locus (+/DAT-cre; +/Tfam.sup.loxP) were identified with PCR. In a
second step such mice were again mated to
Tfam.sup.loxP/Tfam.sup.loxP mice to generate homozygous knockout
animals (+/DAT-cre; Tfam.sup.loxP/Tfam.sup.loxP) and controls
(+/Tfam.sup.loxP or Tfam.sup.loxP/Tfam.sup.loxP).
Southern Blot and PCR Genotyping
[0054] For Southern blot analysis, .about.10 .mu.g of genomic tail
DNA was digested with BamHI, electrophoresed through a 0.8% agarose
gel and transferred to a Hybond C+membrane (Amersham). Membranes
were hybridized with a 3' probe labeled with [.alpha.-.sup.32P]dCTP
(3000 Ci/mmol; Amersham), and visualized by autoradiography using
standard procedures.
[0055] Routine PCR genotyping for the presence of cre was performed
with the forward primer 5'-CAC GAC CAA GTG ACA GCA AT-3' (SEQ ID NO
1) and the reverse primer 5'-AGA GAC GGA AAT CCA TCG CT-3' (SEQ ID
NO 2), giving a product of 371 bp in the presence of cre. PCR
genotyping to verify that the neo gene is excised by FLPe
recombination was done with the forward primer 5'-CCC AAC TTG AGA
TGT ATG AA-3' (SEQ ID NO 3) and the reverse primer 5'-CAA GAA GCG
CTT TAC TGA C-3' (SEQ ID NO 4), giving a product of 954 bp after
excision.
[0056] The Tfam locus was genotyped with PCR using a three primer
strategy (mt31: 5'-CTG CCT TCC TCT AGC CCG GG-3' (SEQ ID NO 5);
mt12: 5'-GTA ACA GCA GAC AAC TTG TG-3' (SEQ ID NO 6); mt36: 5'-CTC
TGA AGC ACA TGG TCA AT-3') (SEQ ID NO 12), generating 437, 404 and
329 bp products for Tfam.sup.loxP, Tfam.sup.wt and Tfam.sup.KO,
respectively (Larsson et al., Nat. Genet. 18, 231-6 (1998)).
In Situ Hybridization
[0057] Probes against the transcripts for tyrosine hydroxylase
(5'-GGT GTG CAG CTC ATC CTG GAC CCC CTC CAA GGA GCG CT-3') (SEQ ID
NO 7) and cre recombinase (5'-GCC CGG ACC GAC GAT GAA GCA TGT TTA
GCT GGC CCA AAT GTT GCT GGA-3'(SEQ ID NO 8) or 5'-CAC CAG AGA CGG
AAA TCC ATC GCT CGA CCA GTT TAG TTA CCC CCA GGC-3') (SEQ ID NO 9)
were 3'-end labeled with .sup.33P and used to detect mtRNA in 14
.mu.m cryostat sections from fresh frozen brains by in situ
hybridization (Dagerlind et al., Histochemistry 98, 39-49. (1992)).
A random oligonucleotide probe was used as a negative control.
Hybridizations were performed at 42.degree. C. for 16-18 hr, and
sections were rinsed five times in 1.times.SSC, dehydrated, and
exposed to photographic emulsion (Kodak NTB2; Eastman Kodak).
Histology and Immunohistochemistry
[0058] Mice were perfused with Ca.sup.2+-free Tyrode's solution,
followed by 4% paraformaldehyde with 0.4% picric acid in 0.16 M
phosphate buffer. The brains were dissected out, post-fixed, and
equilibrated to 10% sucrose containing 0.1% sodium azide. Primary
antibodies used for indirect immunohistochemistry (Hokfelt et al.,
Histochemie 33, 231-54 (1973); Zetterstrom et al., Neuroscience 62,
899-918. (1994)) included polyclonal antibodies against tyrosine
hydroxylase (TH) (1:400; Pel-Freez), .beta.-galactosidase (1:500;
Chemicon), glial fibrillary acidic protein 19 (GFAP-19) (1:500;
Sigma) and .alpha.-synuclein (1:50; Biogenesis). Cryostat sections
(14 .mu.m) were incubated with primary antibodies overnight at
4.degree. C., rinsed, and incubated with appropriate FITC- or
Cy2-labeled secondary antibodies. Controls included omitting the
primary antibody. Sections were analyzed by fluorescence
microscopy.
[0059] For terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end (TUNEL) labeling we used the In situ Cell
Death Detection (POD) kit (Roche) according to the instructions of
the manufacturer.
Behavioral Analyses of Animals
[0060] Open field activity was examined in automated activity
cages. This consisted of Plexiglas boxes (35.times.35.times.18 cm)
with a lower and a higher row of infrared sensitive photocells and
a microcomputer registering interruption of photocell beams.
Animals were tested during the light phase of the light-dark cycle,
between 9:00 and 13:00 h. Locomotor activity was registered when
animals interrupted the lower row of photocells and rearing was
registered when the higher row was interrupted. Animals were
individually placed gently in the open field arena and remained
there during 60 min. Locomotion and rearing counts were registered
every 10 min. For the L-DOPA treatment, spontaneous locomotion and
rearing were measured in activity cages as above. Animals were then
given i.p. injections of L-DOPA combined with the peripheral dopa
decarboxylase inhibitor benzerazide (Madopark 20 mg/kg in PBS;
Roche) or PBS and were then immediately put back into the activity
cages for another 60 minutes of recording. The scoring of counts
was given as mean +/-SEM. Statistical significance was tested using
2-way ANOVA analysis. The open field box was cleaned with 70%
alcohol after each test.
[0061] Locomotor skill and balance were tested using a rotating rod
(Rota-Rod). The mice were given a short practice run in the morning
and then three trials were done in the afternoon with one hour
between every trial. The speed of the rod increased from 4-40 rpm
and each trial was terminated after 300 seconds.
Results
Generation and Breeding of DAT-cre Mice
[0062] Mice with a cell-type specific expression of cre recombinase
in dopaminergic (DA) neurons were generated by knocking in cre at
the dopamine transporter locus (DAT; Solute carrier family 6,
member 3). A targeting vector was constructed spanning 5.5 kb both
upstream and downstream of the endogenous translational start site
located in exon 2. The vector contained a cassette with an NLS-cre
gene followed by an FRT-flanked neomycin resistance (neo) gene
inserted just before the start site in exon 2 to drive cre
expression from the endogenous DAT-promoter (FIG. 1A). The knockin
locus was introduced into embryonic stem (ES) cells by homologous
recombination. After passage through the germline we verified the
presence of both the wild-type (wt) and the knockin
(DAT-cre.sup.neo) allele in the heterozygous mice (FIG. 1B).
[0063] The presence of the neo gene in this targeted locus could
potentially lead to several problems. Neo contains a cryptic splice
site which can cause abnormal splicing and thereby affect the
normal expression of the DAT-locus. Furthermore, the expression of
neo is driven by the thymidine kinase (tk) promoter which may
recruit excess amounts of transcriptional elements to the locus,
thereby causing ectopic expression of cre in non-DA cells. In our
experimental strategy we circumvented this problem by flanking the
neo gene with FRT-sites, enabling us to later remove neo by
crossing the DAT-cre.sup.neo mice to FLPe deleter mice thus
generating DAT-cre mice (FIG. 1A). We demonstrated the excision of
neo both by Southern blot (FIG. 1B) and by PCR (FIG. 1C).
Expression Pattern of cre in Homozygous Knockout Mice
[0064] When DAT-cre.sup.neo and DAT-cre mice were crossed with mice
homozygous for a loxP-flanked Tfam allele
(Tfam.sup.loxP/Tfam.sup.loxP) we only observed unspecific
recombination of the Tfam locus in tail DNA of DAT-cre.sup.neo mice
but not in tail DNA of DAT-cre mice (FIG. 1C). When we repeated the
Tfam genotyping using DNA from heart, kidney, liver, spleen, testis
and cerebral cortex we again saw unspecific recombination of the
Tfam allele in all tissues of DAT-cre.sup.neo, mice but not in any
tissue from DAT-cre mice (data not shown).
[0065] To study the cre expression in the brain we performed in
situ hybridization on brain sections from DAT-cre mice using probes
for both cre and tyrosine hydroxylase (TH), a commonly used marker
for catecholamine neurons. The cre probe gave a clear hybridization
signal in the ventral tegmental area (VTA) and in the substantia
nigra pars compacta (SNpc), overlapping perfectly with the signal
from the TH probe (FIG. 5A), thus demonstrating that the neurons
expressing TH mRNA in the ventral mesencephalon (dopamine neurons)
now co-expressed cre recombinase mRNA. To assess cre activity we
also crossed DAT-cre mice with reporter mice that upon cre mediated
excision of an upstream stop site express the lacZ gene from the
ROSA26 locus (ROSA26-R). Fluorescence immunohistochemistry with
.beta.-gal antibodies showed a highly specific labeling confined to
the DA neurons in the VTA and SNpc area very similar to the
labeling achieved using TH antibodies (FIG. 5B). Thus, also at the
protein level, there was a complete match between neurons
expressing TH immunoreactivity and those expressing .beta.-gal
immunoreactivity.
Generation of Parkinson Mice
[0066] We wanted to generate mice with a parkinsonian phenotype by
knocking out the gene for mitochondrial transcription factor A
(Tfam) in dopaminergic neurons. Tfam has previously been knocked
out in different tissues always resulting in slow and delayed cell
death due to respiratory chain failure. To generate the Parkinson
mice we used a two step breeding protocol. First DAT-cre mice were
crossed to mice homozygous for a loxP-flanked Tfam allele
(Tfam.sup.loxP/Tfam.sup.loxP). DA T-cre; +/Tfam.sup.loxP mice from
this mating were identified and crossed again to
Tfam.sup.loxP/Tfam.sup.loxP mice. This cross generated the four
possible genotypes in approximately Mendelian proportions
(21%+/Tfam.sup.loxP, 24% Tfam.sup.loxP/Tfam.sup.loxP, 27% DAT-cre;
+/Tfam.sup.loxP and 29% DA T-cre; Tfam.sup.loxP/Tfam.sup.loxP).
[0067] DAT-cre; Tfam.sup.loxP/Tfam.sup.loxP pups appeared normal,
developed normally and gained weight as their littermates did (data
not shown). At around 15 weeks of age they started to display
PD-like symptoms such as abnormal gait and bradykinesia. Decreased
locomotion and reduced exploratory behaviour became apparent at
that time. They still appeared to be relatively healthy though,
with normal weight and a well groomed fur. This phenotype remained
constant for several weeks with no further change in behavior or
activity and no weight loss or other signs of decreased health
status. We observed progression of symptoms with tremor, increased
twitching and apparent limb rigidity at about 20 weeks of age. We
provided the mice with moist food on the cage floor from about 20
weeks of age because motor impairment prevented them from reaching
food and water normally provided on the grid covering the cage.
However, even with this procedure, there was a slow gradual decline
of weight as the neurodegeneration progressed (FIG. 2i), and the
homozygous knockout mice had to be euthanized at about 45 weeks of
age because of poor general condition.
Histochemistry of Knockout Brains
[0068] Next, we used histological analyses to characterize the
neuropathology underlying the progressive deterioration of motor
function in homozygous knockout mice (FIG. 3A-N). We performed TH
immunofluorescence in newborn as well as 2 and 6 week old mice and
found that the DA innervation of striatum appeared normal in
homozygous knockout mice at these ages (FIG. 3A-C and data not
shown). We first observed loss of DA nerve terminals in
dorsolateral striatum at age 12 weeks and this loss progressed to
involve most of the dorsal striatum as the homozygous knockout mice
became older (FIG. 3C-F and data not shown). We also observed a
subsequent reduction of DA nerve terminals in ventral striatum,
from 20 weeks of age, which progressed to involve most areas of the
ventral striatum (FIG. 3D-F). The striking end result was an almost
complete loss of TH-immunoreactive nerve terminals (FIGS. 3M,N) in
dorsal as well as ventral striatum, except for parts of nucleus
accumbens, in 43 weeks old homozygous knockout mice (FIG. 3F).
[0069] Cresyl violet staining (not shown) and TH
immunohistochemistry (FIG. 3I-L) revealed progressive loss of DA
neurons in the homozygous knockout mouse midbrain. We observed DA
nerve cell loss in SNpc, which predominantly innervates dorsal
striatum, from age 12 weeks, whereas DA nerve cell loss in VTA,
which predominantly innervates ventral striatum and cortical areas,
was not obvious until 20 weeks of age in homozygous knockout mice
(FIG. 3I-L). There was thus a very good correlation between the
temporal and spatial progression of striatal DA denervation (FIG.
3C-F) and midbrain DA nerve cell loss (FIG. 3I-L).
[0070] Neurochemical studies with HPLC demonstrated a profound
reduction of DA and its metabolites in striatum (caudate putamen
and nucleus accumbens), SNpc, VTA and cortex in 20 weeks old
homozygous knockout mice (FIG. 30-Q). In dorsal striatum DA levels
were reduced to about 1.5% of normal levels. We also found markedly
increased ratios of homovallinic acid (HVA) and
3,4-dihydroxyphenylacetic acid (DOPAC) to DA, indicating an
increased DA turnover in striatum of homozygous knockout mice at
this age (FIG. 3Q). The loss of DA nerve cells and nerve terminals
was thus accompanied by a corresponding loss of DA content in
affected brain regions.
[0071] Many TH positive neurons of homozygous knockout mice
contained rounded TH negative regions in their cytoplasm and we
therefore performed confocal microscopy studies revealing that
these areas were occupied by intracytoplasmic aggregates of
.alpha.-synuclein-immunoreactivity (FIG. 4A-B). We detected small
.alpha.-synuclein-immunoreactive aggregates in many DA neurons in
SNpc and in a few DA neurons in VTA from 6 weeks of age in
homozygous knockout mice. These .alpha.-synuclein aggregates became
larger and more abundant as the homozygous knockout mice aged and
were present in most remaining DA neurons of SNpc and VTA from 16
weeks of age onwards. No .alpha.-synuclein-immunoreactive
inclusions were observed in A12 DA neurons of homozygous knockout
mice (FIG. 4D) or in SNpc, VTA or A12 DA neurons of control mice at
different ages (not shown).
[0072] DA neurons in patients with Parkinson's disease often have
intracytoplasmic Lewy bodies, which contain ubiquitin-conjugated
.alpha.-synuclein aggregates (Dauer et al., Neuron 39, 889-909
(2003)). We therefore performed additional confocal microscopy and
found that the .alpha.-synuclein-immunoreactive aggregates were not
immunoreactive for ubiquitin in younger homozygous knockout mice
(not shown). However, a subset of the .alpha.-synuclein aggregates
was also immunoreactive for ubiquitin in homozygous knockout mice
older than 20 weeks (FIG. 4C).
[0073] TUNEL labeling did not reveal any DA neurons undergoing
apoptotic death in mesencephalon of homozygous knockout mice at
different ages (not shown). We also performed immunohistochemistry
to detect glial fibrillary acidic protein (GFAP) and found no
significantly increased expression in striatum and a very slight
increase in SNpc (not shown) of homozygous knockout mice. These
observations suggest that the cell death in SNpc and VTA of
homozygous knockout mice is a continuous and slowly progressive
process that is not associated with any marked astroglial
response.
Behavioral Analysis of Parkinson Mice
[0074] We performed behavioral tests of homozygous knockout and
control mice of different ages to obtain quantitative measurements
of the clinically obvious motor impairment. We found a trend
towards lower counts for both locomotion (FIG. 2A) and rearing
(FIG. 2D) in 10 week old homozygous knockout mice, but these
differences were not statistically significant. However, we found a
clear reduction of both locomotion (FIG. 2B) and rearing (FIG. 2E)
in 14 week old homozygous knockout mice and both of these
activities declined progressively as homozygous knockout mice grew
older (FIGS. 2C,F). We also induced motor activity with the
Rota-Rod and found a trend towards decreased performance in 10 week
old homozygous knockout mice (FIG. 2G) and a significant reduction
in 14 week old homozygous knockout mice (FIG. 2H). Control mice
typically improved their performance on the Rota-Rod as the test
was repeated (FIGS. 2G,H). Most of the 14 week old control mice
remained on the rod for the full 300 seconds of the experiment
during the third trial, whereas homozygous knockout mice of the
same age were unable to improve their locomotor performance (FIG.
2H). Strikingly, the homozygous knockout mice were unable to move
for an extended period of time after that they had fallen off the
Rota-Rod, whereas control mice immediately started to explore the
new surroundings.
[0075] We used a standard Parkinson's disease drug containing
L-DOPA (Madopark, Roche) to treat 20 and 30 week old homozygous
knockout mice and observed improved motor performance (FIG. 4E-H).
Interestingly, an identical L-DOPA dose elicited a more pronounced
and longer lasting locomotion response in the younger homozygous
knockout mice (FIGS. 4E,F), whereas the increase in rearing was
similar in homozygous knockout mice at both ages (FIGS. 4G,H). The
locomotor response abruptly terminated in the older homozygous
knockout mice resembling the "wearing off" effect seen
clinically.
[0076] The foregoing disclosure has been set forth merely to
illustrate the invention and is not intended to be limiting. Since
modifications of the disclosed embodiments incorporating the spirit
and substance of the invention may occur to persons skilled in the
art, the invention should be construed to include everything within
the scope of the appended claims and equivalents thereof. Patent
applications, patents and literature references cited herein
indicate the knowledge in this field and are hereby incorporated by
reference in their entirety. Where inconsistent interpretations are
possible, the disclosure herein controls.
Sequence CWU 1
1
12148DNAArtificialsynthetic probe 1caccagagac ggaaatccat cgctcgacca
gtttagttac ccccagcc 48220DNAArtificialsynthetic probe 2agagacggaa
atccatcgct 20320DNAArtificialsynthetic probe 3cccaacttga gatgtatgaa
20419DNAArtificialsynthetic probe 4caagaagcgc tttactgac
19520DNAArtificialsynthetic probe 5ctgccttcct ctagcccggg
20620DNAArtificialsynthetic probe 6gtaacagcag acaacttgtg
20738DNAArtificialsynthetic probe 7ggtgtgcagc tcatcctgga ccccctccaa
ggagcgct 38848DNAArtificialsynthetic probe 8gcccggaccg acgatgaagc
atgtttagct ggcccaaatg ttgctgga 48920DNAArtificialsynthetic probe
9cacgaccaag tgacagcaat 201020DNAArtificialsynthetic probe
10ctctgaagca catggtcaat 201122DNAArtificialsynthetic probe
11atgaaccagg tcttgagtct gg 221222DNAArtificialsynthetic probe
12cagggtcgga gagtcataca ac 22
* * * * *